1. Field of the Invention
The present invention relates generally to the field of transgenic animals. More particularly, it concerns methods for generating primordial germ cell-derived cell lines, transforming primordial germ cells and primordial germ cell-derived cell lines, and using these transformed cells and cell lines to generate transgenic non-rodent animal species.
2. Description of Related Art
Animals having certain desired traits or characteristics, such as increased weight gain, feed efficiency, carcass composition, milk production or content and disease resistance have long been desired. Traditional breeding processes are capable of producing animals with some desired traits, but these are often accompanied by a number of undesired characteristics, and is an extremely costly and time consuming process.
The development of transgenic animal technology holds great promise for the production of animals having specific, desired traits. Transgenic animals are animals that carry a gene that has been deliberately introduced into somatic and germline cells at an early stage of development. Although transgenic animals have been produced by various methods in several different species, methods to readily and reproducibly produce transgenic large mammals at reasonable costs are still lacking.
At present the only techniques available for the generation of transgenic domestic animals are by pronuclear injection or use of viral vectors. In both cases the incoming DNA inserts at random, which can cause a variety of problems. The first of these problems is insertional inactivation, which is inactivation of an essential gene due to disruption of the coding or regulatory sequences by the incoming DNA. Another problem is that the transgene may either be not incorporated at all, or incorporated but not expressed. A further problem is the possibility of inaccurate regulation due to positional effects. This refers to the variability in the level of gene expression and the accuracy of gene regulation between different founder animals produced with the same transgenic constructs. Thus, it is not uncommon to generate 10 founder animals and identify only one that expresses the transgene in a manner that warrants the maintenance of the transgenic line.
Additionally, using the present technology, it is not possible to fully inactivate or remove genes in transgenic animals, only add new genes. As a result it is not possible to delete genes involved in undesired cellular processes, or to undertake any genetic modification that entails changes in existing genes. Moreover, the efficiency of generating transgenic domestic animals is low, with efficiencies of 1 in 100 offspring generated being transgenic not uncommon (Wall, 1996). As a result the cost associated with generation of transgenic animals can be as much as 250-500 thousand dollars per expressing animal (Wall, 1996).
These drawbacks are overcome by the utilization of homologous recombination (Koller and Smithies, 1992), which directs the insertion of the transgene to a specific location. This technique allows the precise modification of existing genes, and overcomes the problems of positional effects and insertional inactivation. Additionally, it allows the inactivation of specific genes as well as the replacement of one gene for another. Unfortunately the efficiency of the procedure is so low that it cannot be utilized directly on embryos but must make use of a carrier cell line. The availability of appropriate cell lines will allow the precise manipulation of the genomic material followed by the generation of a living animal carrying those changes.
Embryonic stem (ES) cells, isolated from the inner cell mass (ICM) of the preimplantation embryo, possess the ability to proliferate indefinitely in an undifferentiated state, and are capable of contributing to the formation of normal tissues and organs of a chimeric individual when injected into a host embryo. The ES cell line allows manipulation and selection in vitro, followed by the generation of a transgenic animal carrying those changes. The ability to colonize the germ line following culture and genetic manipulation have made ES cells a powerful tool for the modification of the genome in the mouse species. Chimeras produced between genetically modified ES cells and normal embryos have been used to study in vivo gene regulation (Stewart et al., 1985), as well as germ-line transmission of introduced genes (Smithies 1991). In addition, ES cells have been used to study targeted modification of genes by homologous recombination (Smithies 1991).
The use of chimeras has been shown to be effective in producing transgenic mice. About 70% of expanded mouse blastocysts develop into live young with about 50% of the young born being chimeric (Bradley et al., 1984). Twenty percent of these chimeric young have germ cell chimerism. Utilizing this method it is possible that chimerism in the germ line may be 20-30%. However, the ES-cell method has not been successfully applied to production of larger transgenic mammals, for example, transgenic pigs, cattle, goats or sheep. A reason for the failure to extrapolate methods from mice to larger mammals may be the difference in developmental stages of the species (Wheeler, 1996).
Recently, it has been reported that murine cell lines derived from primordial germ cells (PGC) behave similarly to ES cells and are capable of contributing to the germ line (Labosky et al., 1994). These cells, referred to as embryonic germ (EG) cells or PGC-derived cells (Labosky et al., 1994; Strelchenko, 1996), are indistinguishable from ES cells in terms of markers of the undifferentiated state as well as their ability to colonize the germ line following injection into host blastocysts (Labosky et al., 1994; Stewart et al, 1994). Thus, even though the starting tissue source or cellular phenotype differ from the ICM-derived cell lines, once established they have similar, if not identical, properties.
Although the majority of the research on ES and primordial germ cells has been done in the mouse, attempts at developing this technology in other mammalian species have been reported. Embryonic cell lines have been described from hamster (Doetschman et al. 1988), mink (Sukoyan et al., 1992, 1993), rabbit (Graves and Moreadith, 1993; Giles et al., 1993), pig (Piedrahita et al., 1990; Strojek et al., 1990; Notarianni et al., 1990; Talbot et al., 1993; Wheeler, 1994; Gerfen and Wheeler, 1995; Shim and Anderson, 1995), sheep (Handyside et al., 1987; Piedrahita et al., 1990; Notarianni et al, 1991; Campbell et al., 1995) and cattle (Saito et al., 1992; Sims and First, 1993; Stice et al, 1994; Strelchenko, 1996; Stice and Strelchenko, 1996). Although each of these cell lines have some of the characteristics of the ES cells described from mice, germ line transmission, a prerequisite for generation of a transgenic line of animals, has not been demonstrated.
Another problem associated with the generation of transgenic animals is the difficulty with transformation of ES or EG cells with DNA carrying a desired trait or traits. These difficulties are related to the inability of the cells to remain unchanged (undifferentiated) upon repeated passage. This is in contrast with mouse ES cells, which can be passaged multiple times without major changes in the potential to generate a transgenic animal. To date there have been no reports on the generation of undifferentiated transformed transgenic cell lines of embryo-derived or PGC-derived cell lines in any non-rodent domestic animal species.
A genetically transformed ES or PGC-derived cell line capable of taking part in chimera formation, or nuclear transfer development in enucleated oocytes, would be of great value for the medical, veterinary, and agricultural community. In the medical and veterinary field it would allow the generation of biopharmaceuticals and oral immunogens in the milk, the generation of animals that can be used as human tissue donors, the development of animal models of human disease that can speed the development of alternative therapeutic methods including gene therapy, and the development of blood substitutes. In the veterinary field it would allow the generation of animals that are naturally immune to particular diseases. In the agricultural field it will allow the modification of the milk composition to increase shelf life, cheese yield, and permit lactose intolerant individuals to safely consume the modified milk. It will also allow the introduction of small genetic changes that can modify disease resistance, growth rate and carcass composition, wool composition, and nutritional efficiency, among others. Unfortunately, to date, there has been no description of transformed non-rodent ES or PGC-derived cell lines.
The present invention overcomes the problems in generating non-rodent transgenic animals described in the art by providing methods for the isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, methods for transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated by the present invention is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.
Accordingly, the present invention provides a method of growing primordial germ cells from a non-rodent animal species comprising plating the primordial germ cells on feeder cells, the feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor. In certain aspects, the method may comprise plating the primordial germ cells on feeder cells, the feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, for an amount of time sufficient to obtain an undifferentiated primordial germ cell colony.
In an alternative aspect, the invention provides a method of growing primordial germ cells from a non-rodent animal species, comprising plating a composition comprising primordial germ cells from an embryo of said non-rodent animal species on STO feeder cells, said STO feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor.
In yet another aspect, the invention provides a method of growing primordial germ cells from a non-rodent animal species, comprising plating a composition comprising primordial germ cells from an embryo of said non-rodent animal species on feeder cells, said feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor.
In a further aspect, the invention provides a method of growing primordial germ cells from a non-rodent animal species, comprising plating a composition comprising primordial germ cells from an embryo of said non-rodent animal species on feeder cells, said feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added leukemia inhibitory factor.
In a further aspect, the invention provides a method of growing primordial germ cells from a non-rodent animal species, comprising plating a composition comprising primordial germ cells from an embryo of said non-rodent animal species on feeder cells, said feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor or leukemia inhibitory factor.
Additionally, the invention provides a method of growing primordial germ cells from a non-rodent animal species, comprising plating a composition comprising primordial germ cells from an embryo of said non-rodent animal species on feeder cells other than S1/S14 or S1-m220, said feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor.
The invention also provides a method of growing primordial germ cells from a non-rodent animal species, comprising plating a composition comprising primordial germ cells from an embryo of said non-rodent animal species on feeder cells other than S1/S14 or S1-m220, said feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor or leukemia inhibitory factor
In certain preferred embodiments, the composition comprising the primordial germ cells is isolated from an embryo of the non-rodent animal species by the steps of collecting an embryo of a non-rodent animal species, removing the genital ridge from the embryo, incubating the genital ridge in a biologically acceptable solution, disrupting the genital ridge thereby releasing the primordial germ cells, and collecting the primordial germ cells to provide the composition comprising the primordial germ cells. In certain embodiments, the primordial germ cells are collected by centrifugation.
In particular aspects of the present invention, the primordial germ cells comprise at least a first exogenous DNA segment. Primordial germ cells comprising exogenous DNA are referred to as genetically transformed primordial germ cells. In further embodiments, the primordial germ cells are provided with an exogenous, selected DNA segment by electroporation, particle bombardment or calcium phosphate precipitation. In certain aspects of the invention the composition comprising primordial germ cells is provided with a selected DNA segment and the primordial germ cells that contain the selected DNA segment are selected and optionally separated away from the primordial germ cells of the composition that do not contain the selected DNA segment.
The density of the feeder cells is critical to the success of a number of the methods described herein. Within the range of densities of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, the actual density may vary, depending on the particular application. Therefore, in certain aspects of the present invention, the density of the feeder cells may be between about 1.5xc3x97105 cells/cm2 and about 5xc3x97105 cells/cm2, between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, between about 2xc3x97105 cells/cm2 and about 9xc3x97105 cells/cm2, between about 3xc3x97105 cells/cm2 and about 8xc3x97105 cells/cm2, between about 2xc3x97105 cells/cm2 and about 5xc3x97105 cells/cm2, between about 4xc3x97105 cells/cm2 and about 7xc3x97105 cells/cm2, between about 2.5xc3x97105 cells/cm2 and about 7.5xc3x97105 cells/cm2, between about 5xc3x97105 cells/cm2 and about 8xc3x97105 cells/cm2, between about 1.5xc3x97105 cells/cm2 and about 3xc3x97105 cells/cm2, between about 5xc3x97105 cells/cm2 and about 6xc3x97105 cells/cm2, between about 4xc3x97105 cells/cm2 and about 6.5xc3x97105 cells/cm2, between about 5xc3x97105 cells/cm2 and about 1xc3x97106 cells/cm2, between about 8xc3x97105 cells/cm2 and about 9xc3x97105 cells/cm2, between about 2.5xc3x97105 cells/cm2 and about 5xc3x97105 cells/cm2, or any combination of densities within the range.
Thus for a particular embodiment, the density of the stock of feeder cells may be about 1.5xc3x97105 cells/cm2, 2xc3x97105 cells/cm2, 2.5xc3x97105 cells/cm2, 3xc3x97105 cells/cm2, 4xc3x97105 cells/cm2, 5xc3x97105 cells/cm2, 6xc3x97105 cells/cm2, 7xc3x97105 cells/cm2, 7.5xc3x97105 cells/cm2, 8xc3x97105 cells/cm2, 9xc3x97105 cells/cm2, or 1xc3x97106 cells/cm2. Another way of expressing the density of the feeder cells is by calculating the number of feeder cells used per 35 mm well. Thus, densities of feeder cells of between about 1 million and about 9 million or so per 35 mm well are preferred for use in the present invention. Thus the density of feeder cells may be about 1 million, about 1.5 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 7.5 million, about 8 million, about 8.5 million or about 9 million per 35 mm well, with about 3 million or so per 35 mm well being particularly preferred in certain aspects.
The isolated composition comprising the primordial germ cells is grown on a layer of feeder cells. The feeder cells provide a microenvironment conducive to the growth of the primordial germ cells. The feeder cells provide growth factors to the growing primordial germ cells, as well as providing an extracellular matrix. In certain aspects of the present invention, the feeder cell lines may be engineered to express selected growth factors. Thus in certain embodiments of the present invention, the feeder cells may comprise at least a first exogenous DNA sequence. Exemplary types of feeder cells preferred for use in the present invention are embryonic cell lines such as embryonic fibroblasts from selected animal species, such as murine, porcine or bovine. In certain aspects of the present invention, the feeder cells may be murine Sl/Sl4 cells. In other embodiments, the feeder cells may be STO cells (mouse embryonic fibroblast cells), while in other particular aspects, the feeder cells may be Sl4-m220 cells. Mixed cultures of cells are also contemplated for use as feeder cells in certain aspects of the invention. Thus, in further aspects of the present invention, the feeder cells comprise at least a first cell type and at least a second distinct cell type. In certain aspects, the feeder cells are a mixture of STO and porcine embryonic fibroblasts.
The feeder cells are inactivated prior to use, preferably by X-irradiation or using mitomycin C. In preferred embodiments of the present invention, the feeder cells are inactivated with cobalt radiation or cesium radiation.
The present invention also provides for culturing the isolated primordial germ cells in an appropriate medium. As discussed above, the feeder cells provide growth factors to the growing primordial germ cells, however, the amount of endogenous growth factors provided may vary from preparation to preparation of feeder cells. Therefore, in certain aspects of the invention exogenously added growth factors may be added to supplement the endogenous supply.
A growth factor that is critical for growth of the primordial germ cells of the present invention is basic fibroblast growth factor. As is the case with each of the growth factors described herein, basic fibroblast growth factor can be utilized from a variety of mammalian sources, including, but not limited to, porcine, bovine, ovine, caprine, equine, murine or human. In particular aspects human basic fibroblast growth factor is preferred. In certain aspects the growth factors, such as basic fibroblast growth factor, is from the same species as the primordial germ cells, or in other aspects from a different species as the primordial germ cells.
In preferred embodiments, the culture medium may comprise human basic fibroblast growth factor at a concentration of between about 5 ng/ml and about 100 xcexcg/ml. In more preferred embodiments, the medium comprises human basic fibroblast growth factor at a concentration of about 40 ng/ml. However, it will be understood that the range of concentrations may be between about 5 ng/ml and about 10 xcexcg/ml, or between about 10 ng/ml and about 100 xcexcg/ml. Equally, the range can be between about 10 ng/ml and about 50 xcexcg/ml, between about 10 ng/ml and about 1 xcexcg/ml or between about 20 ng/ml and about 250 ng/ml.
It is also understood that about 5 ng/ml includes about 6 ng/ml, about 7 ng/ml, about 8 ng/ml and the like, while about 100 xcexcg/ml includes about 99 xcexcg/ml, about 98 xcexcg/ml, about 97 xcexcg/ml and the like. Additionally, the values at the lower end of the range can be lower than the value provided, for example about 4 ng/ml, about 3 ng/ml and still be within the scope of the present invention. Similarly, the upper end of the range includes values such as about 101 xcexcg/ml, about 102 xcexcg/ml within the scope of the present invention. Optimization of the concentration of these or any other of the media components described below can be performed by those of skill in the art without undue experimentation, by testing different concentrations and measuring the effect on growth of primordial germ cell-derived colonies.
In certain aspects of the invention, other members of the fibroblast growth factor family may be used in addition to basic fibroblast growth factor. These members include, but are not limited to, FGF-1 (acidic fibroblast growth factor), FGF-3 (int-2), FGF-4 (hst/K-FGF), FGF-5, FGF-6, and FGF-7.
Other growth factors may be added to the medium in an amount effective to improve the growth characteristics of the primordial germ cells, or to help maintain the primordial germ cells in an undifferentiated state. Thus, in particular embodiments, the culture medium may also comprise an effective amount of leukemia inhibitory factor. In certain aspects, the culture medium comprises leukemia inhibitory factor at a concentration of between about 5 ng/ml and about 100 xcexcg/ml. In more preferred embodiments, the culture medium comprises leukemia inhibitory factor at a concentration of between about 10 ng/ml and about 10 xcexcg/ml. In more preferred embodiments, the culture medium comprises leukemia inhibitory factor at a concentration of between about 15 ng/ml and about 1 xcexcg/ml. In especially preferred embodiments, the culture medium comprises leukemia inhibitory factor at a concentration of about 20 ng/ml. However, it will be understood that the range of concentrations may be between about 5 ng/ml and about 10 xcexcg/ml, or between about 10 ng/ml and about 100 xcexcg/ml. Equally, the range can be between about 10 ng/ml and about 50 xcexcg/ml, between about 10 ng/ml and about 1 xcexcg/ml or between about 20 ng/ml and about 250 ng/ml.
It is also understood that about 5 ng/ml includes about 6 ng/ml, about 7 ng/ml, about 8 ng/ml and the like, while about 100 xcexcg/ml includes about 99 xcexcg/ml, about 98 xcexcg/ml, about 97 xcexcg/ml and the like. Additionally, the values at the lower end of the range can be lower than the value provided, for example about 4 ng/ml or about 3 ng/ml and still be within the scope of the present invention. Similarly, the upper end of the range includes values such as about 101 xcexcg/ml or about 102 xcexcg/ml within the scope of the present invention.
In other embodiments, the culture medium may also comprise an effective amount of uteroferrin. In certain embodiments, the culture medium comprises uteroferrin at a concentration of between about 1 ng/ml and about 100 xcexcg/ml. In certain embodiments, the culture medium comprises uteroferrin at a concentration of between about 10 ng/ml and about 5 xcexcg/ml or between about 20 ng/ml and about 500 ng/ml. In certain aspects of the present invention, the culture medium comprises uteroferrin at a concentration of about 40 ng/ml. In additional embodiments, the culture medium may also comprise an effective amount of soluble stem cell factor. In particular aspects, the culture medium comprises soluble stem cell factor at a concentration of between about 1 ng/ml and about 100 xcexcg/ml. In other embodiments, the culture medium comprises soluble stem cell factor at a concentration of between about 10 ng/ml and about 5 xcexcg/ml. In still more preferred embodiments, the culture medium comprises soluble stem cell factor at a concentration of between about 20 ng/ml and about 250 ng/ml. In exemplary embodiments, the culture medium comprises soluble stem cell factor at a concentration of about 40 ng/ml. However, it will be understood that the range of concentrations of these factors can be between about 1 ng/ml and about 10 xcexcg/ml, or between about 10 ng/ml and about 100 xcexcg/ml. Equally, the range can be between about 10 ng/ml and about 50 xcexcg/ml, between about 10 ng/ml and about 1 xcexcg/ml or between about 20 ng/ml and about 250 ng/ml.
It will be understood by those of skill in the art that about 1 ng/ml includes about 2 ng/ml, about 3 ng/ml, about 4 ng/ml and the like, while about 100 xcexcg/ml includes about 99 xcexcg/ml, about 98 xcexcg/ml, about 97 xcexcg/ml and the like. Additionally, the values at the lower end of the range can be lower than the value provided, for example about 0.8 ng/ml, about 0.5 ng/ml and still be within the scope of the present invention. Similarly, the upper end of the range includes values such as about 101 xcexcg/ml, about 102 xcexcg/ml within the scope of the present invention.
In further embodiments, the culture medium may also comprise an effective amount of xcex12-macroglobulin. In particular aspects, the culture medium comprises xcex12-macroglobulin at a concentration of between about 10 ng/ml and about 10 xcexcg/ml. In other embodiments, the culture medium comprises soluble stem cell factor at a concentration of between about 50 ng/ml and about 5 xcexcg/ml. In still more preferred embodiments, the culture medium comprises soluble stem cell factor at a concentration of between about 100 ng/ml and about 2.5 xcexcg/ml. In exemplary embodiments, the culture medium comprises soluble stem cell factor at a concentration of about 1 xcexcg/ml. However, it will be understood that the range of concentrations of these factors can be between about 10 ng/ml and about 2.5 xcexcg/ml, between about 100 ng/ml and about 10 xcexcg/ml. Equally, the range can be between about 100 ng/ml and about 5 xcexcg/ml, between about 250 ng/ml and about 2.5 xcexcg/ml or between about 500 ng/ml and about 1 xcexcg/ml.
It will be understood by those of skill in the art that about 10 ng/ml includes about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml and the like, while about 10 xcexcg/ml includes about 9 xcexcg/ml, about 8 xcexcg/ml, about 7 xcexcg/ml and the like. Additionally, the values at the lower end of the range can be lower than the value provided, for example about 8 ng/ml, about 5 ng/ml and still be within the scope of the present invention. Similarly, the upper end of the range includes values such as about 11 xcexcg/ml or about 12 xcexcg/ml within the scope of the present invention.
The present invention provides certain embodiments wherein the culture medium may also comprise an effective amount amino acids non-essential to the particular non-rodent animal. In further embodiments, the culture medium comprises amino acids non-essential to the particular non-rodent animal at a concentration of between about 10 nM and about 250 nM. In additional aspects, the culture medium comprises amino acids non-essential to the particular non-rodent animal at a concentration of between about 50 nM and about 150 nM. In still other embodiments, the culture medium comprises amino acids non-essential to the particular non-rodent animal at a concentration of about 100 nM. However, it will be understood that the range of concentrations can be between about 10 nM and about 100 nM, or between about 20 nM and about 250 nM. Equally, the range can be between about 20 nM and about 150 nM, between about 50 nM and about 125 nM or between about 75 nM and about 110 nM.
It is also understood that about 10 nM includes about 11 nM, about 12 nM, about 13 nM and the like, while about 250 nM includes about 249 nM, about 248 nM, about 247 nM and the like. Additionally, the values at the lower end of the range can be lower than the value provided, for example about 9 nM, about 8 nM and still be within the scope of the present invention. Similarly, the upper end of the range includes values such as about 251 nM or about 252 nM that fall within the scope of the present invention.
In preferred embodiments of the present invention, the culture medium may also comprise an effective amount of L-glutamine. In particular aspects, the culture medium comprises L-glutamine at a concentration of between about 0.1 mM and about 50 mM. In more preferred embodiments, the culture medium comprises L-glutamine at a concentration of between about 1 mM and about 20 mM. In still more preferred embodiments, the culture medium comprises L-glutamine at a concentration of about 2 mM. However, it will be understood that the range of concentrations can be between about 0. 1 mM and about 10 mM, or between about 0.5 mM and about 50 mM. Equally, the range can be between about 0.7 mM and about 10 mM, between about 1 mM and about 5 mM or between about 1.5 mM and about 2.5 mM.
It is also understood that about 0.1 mM includes about 0.2 mM, about 0.3 mM, about 0.4 mM and the like, while about 50 mM includes about 49 mM, about 48 mM, about 47 mM and the like. Additionally, the values at the lower end of the range can be lower than the value provided, for example about 0.09 mM or about 0.08 mM and still be within the scope of the present invention. Similarly, the upper end of the range includes values such as about 51 mM or about 52 mM that still fall within the scope of the present invention.
In other preferred embodiments of the present invention, the culture medium may also comprise an effective amount of xcex2-mercaptoethanol. In certain aspects, the culture medium comprises xcex2-mercaptoethanol at a concentration of between about 1 xcexcM and about 1 mM. In further embodiments, the culture medium comprises xcex2-mercaptoethanol at a concentration of between about 25 xcexcM and about 250 xcexcM. In exemplary embodiments, the culture medium comprises xcex2-mercaptoethanol at a concentration of about 100 xcexcM. However, it will be understood that the range of concentrations can be between about 1 xcexcM and about 500 xcexcM, or between about 5 xcexcM and about 1 xcexcM. Equally, the range can be between about 20 xcexcM and about 250 xcexcM, between about 50 xcexcM and about 125 xcexcM or between about 75 xcexcM and about 10 xcexcM.
It is also understood that about 1 xcexcM includes about 0.9 xcexcM, about 0.8 xcexcM, about 0.7 xcexcM and the like, while about 1 mM includes about 2 mM, about 3 mM, about 4 mM and the like. Additionally, the values at the lower end of the range can be lower than the value provided, for example about 0.9 xcexcM or about 0.8 xcexcM and still be within the scope of the present invention. Similarly, the upper end of the range includes values such as about 2 mM or about 3 mM within the scope of the present invention. As discussed above, optimization of the concentration of this or other media components can be performed by those of skill in the art without undue experimentation by testing different concentrations and measuring the effect on growth of primordial germ cell-derived colonies.
In certain embodiments, the culture medium may also comprise an effective amount of Dulbecco""s modified Eagle""s media. The Dulbecco""s modified Eagle""s media may be either low sodium Dulbecco""s modified Eagle""s media or high sodium Dulbecco""s modified Eagle""s media. In exemplary embodiments, the culture medium comprises Dulbecco""s modified Eagle""s media at about 50% volume/volume. In other embodiments, the culture medium may also comprise Ham""s F10 media. In more preferred embodiments, the culture medium comprises Ham""s F10 media at about 50% volume/volume. In exemplary embodiments of the present invention, the culture medium comprises Dulbecco""s modified Eagle""s media at about 50% volume/volume and Ham""s F10 media at about 50% volume/volume. It is understood that the amount of Dulbecco""s modified Eagle""s media or Ham""s F10 media can be about 40% volume/volume, about 30% volume/volume and the like. Additionally, about 50% volume/volume includes about 49%, about 48%, and the like, as well as about 51%, about 52% and about 53% while remaining within the scope of the invention.
Culture media comprising combinations of different growth factors are also contemplated for use in the present invention. Thus, in certain aspects of the present invention, the culture medium comprises an effective amount of basic fibroblast growth factor and an effective amount of at least one of uteroferrin, xcex12-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential to said non-rodent animal, L-glutamine, xcex2-mercaptoethanol, Dulbecco""s modified Eagle""s media or Ham""s F10 media. In further aspects, the culture medium comprises an effective amount of basic fibroblast growth factor and a combined effective amount of at least two of uteroferrin, xcex12-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential to said non-rodent animal, L-glutamine, xcex2-mercaptoethanol, Dulbecco""s modified Eagle""s media or Ham""s F10 media.
In preferred aspects of the present invention, the culture medium comprises an effective amount of basic fibroblast growth factor and a combined effective amount of at least three of uteroferrin, xcex12-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential to said non-rodent animal, L-glutamine, xcex2-mercaptoethanol, Dulbecco""s modified Eagle""s media or Ham""s F 10 media. In further aspects of the present invention, the culture medium comprises an effective amount of basic fibroblast growth factor and a combined effective amount of uteroferrin, xcex12-macroglobulin and leukemia inhibitory factor. In particular embodiments, the culture medium comprises basic fibroblast growth factor at a concentration of between about 5 ng/ml and about 100 xcexcg/ml, uteroferrin at a concentration of between about 1 ng/ml and about 100 xcexcg/ml, xcex12-macroglobulin at a concentration of between about 10 ng/ml and about 10 xcexcg/ml and leukemia inhibitory factor at a concentration of between about 5 ng/ml and about 100 xcexcg/ml.
In certain embodiments of the present invention, the medium comprises between about 5 ng/ml and about 100 xcexcg/ml of basic fibroblast growth factor, between about 1 ng/ml and about 100 xcexcg/ml of uteroferrin, between about 10 ng/ml and about 10 xcexcg/ml of xcex12-macroglobulin, between about 5 ng/ml and about 100 xcexcg/ml of leukemia inhibitory factor, between about 1 ng/ml and about 100 xcexcg/ml of soluble stem cell factor, between about 10 nM and about 250 nM of non-essential amino acids, between about 0.1 mM and about 50 mM of L-glutamine, between about 1 xcexcM and about 1 mM of xcex2-mercaptoethanol, about 50% volume/volume of Dulbecco""s modified Eagle""s media, and about 50% volume/volume of Ham""s F10 media.
In exemplary embodiments of the present invention, the medium comprises about 40 ng/ml of basic fibroblast growth factor, about 40 ng/ml of uteroferrin, about 1 xcexcg/ml of xcex12-macroglobulin, about 20 ng/ml of leukemia inhibitory factor, about 40 ng/ml of soluble stem cell factor, about 100 nM of non-essential amino acids, about 2 mM of L-glutamine, about 0.1 mM of xcex2-mercaptoethanol, about 50% volume/volume of Dulbecco""s modified Eagle""s media, and about 50% volume/volume of Ham""s F10 media.
The instant invention also provides methods wherein the plated primordial germ cells are maintained in an undifferentiated state for about 2 passages, about 3 passages, about 4 passages, about 5 passages, about 6 passages, about 7 passages, about 8 passages, about 9 passages, about 10 passages, about 11 passages, about 12 passages, about 13 passages or about 14 passages. In other embodiments of the present invention, the plated primordial germ are maintained in an undifferentiated state for about 20 passages, about 30 passages, about 50 passages or about 100 passages.
As used herein, the term xe2x80x9cnon-rodent animalxe2x80x9d will be understood to include all vertebrate animals, except rodents and humans. In certain embodiments of the present invention, the non-rodent animal species is bovine. In other embodiments, the non-rodent animal species is ovine. In still other embodiments, the non-rodent animal species is porcine. In yet other embodiments, the non-rodent animal species is caprine. Other non-rodent animals contemplated for use in the present invention include, but are not limited to, horses (equine), buffaloes and rabbits.
The present invention provides a primordial germ cell from a non-rodent animal species that may be prepared by a process comprising plating a composition comprising primordial germ cells on a stock of feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor. In particular embodiments, the invention provides a primordial germ cell colony from a non-rodent animal species that may be prepared by a process comprising the steps of plating a composition comprising primordial germ cells on a stock of feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor for an amount of time sufficient to obtain a primordial germ cell colony. In preferred aspects, the primordial germ cell colony is in an undifferentiated state.
Additionally, the present invention provides a method of preparing a primordial germ cell-derived cell line from a non-rodent animal species, that may comprise plating a composition comprising primordial germ cells on a stock of feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, and culturing the plated primordial germ cells for a period of time effective to provide a primordial germ cell-derived cell line.
Thus, the instant invention provides a primordial germ cell-derived cell line from a non-rodent animal species, that may be prepared by a process comprising plating a composition comprising primordial germ cells on a stock of feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, and culturing the plated primordial germ cells for an effective period of time to provide a primordial germ cell-derived cell line.
Additionally, the present invention provides a method of preparing primordial germ cells of a non-rodent animal species that contain a selected DNA segment, that may comprise introducing a selected DNA segment into a composition comprising primordial germ cells from the non-rodent animal species to obtain candidate primordial germ cells that contain the selected DNA segment, and plating the candidate primordial germ cells of the non-rodent animal species that contain the selected DNA segment on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain the primordial germ cells of the non-rodent animal species that contain the selected DNA segment.
In particular aspects, the method may comprise the steps of introducing a selected DNA segment into a composition comprising primordial germ cells from the non-rodent animal species to obtain candidate primordial germ cells that contain the selected DNA segment, screening said candidate primordial germ cells of the non-rodent animal species for the presence of the selected DNA segment, and plating the candidate primordial germ cells of the non-rodent animal species that contain the selected DNA segment on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, for an amount of time sufficient to obtain a colony comprising the primordial germ cells of the non-rodent animal species that contain the selected DNA segment.
In further aspects of the present invention, the method may comprise the steps of plating a composition comprising primordial germ cells on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, for a period of time sufficient to obtain at least a first passage, introducing a selected DNA segment into the composition comprising primordial germ cells from the non-rodent animal species to obtain candidate primordial germ cells that contain the selected DNA segment, and plating the candidate primordial germ cells of the non-rodent animal species that contain the selected DNA segment on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain the primordial germ cells of the non-rodent animal species that contain the selected DNA segment.
In particular methods of the present invention, the primordial germ cells of the non-rodent animal species that contain the selected DNA segment are cultured for between about 2 and about 14 passages. In other preferred methods, the primordial germ cells of the non-rodent animal species that contain the selected DNA segment are cultured for about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 or about 13 passages.
In exemplary methods of the present invention, the selected DNA segment is introduced into the primordial germ cell by electroporation. In other methods, the selected DNA segment is introduced into the primordial germ cell by particle bombardment, calcium phosphate transformation or by viral transformation.
In certain embodiments, the selected DNA segment may comprise at least a first coding region encoding a selected protein, wherein the coding region is expressed in one or more of the primordial germ cells. In further embodiments, the first coding region encodes a selected disease resistance, carcass composition, weight gain, coat composition or milk component protein. In other embodiments, the first coding region encodes a selected marker protein. In exemplary embodiments, the first coding region encodes green fluorescent protein that has been adapted to increase expression in the non-rodent animal species. A protein is xe2x80x9cadapted to increase expression inxe2x80x9d a non-rodent animal species by altering the coding sequence of the protein to use codons that are preferred for use in the particular non-rodent animal species desired for use. In still other embodiments, the first coding region encodes a neomycin resistance protein. In further embodiments, the first coding region encodes GP63, myelin basic protein, hCD59, Factor IX, xcex1-antitrypsin, xcex1-casein, an interleukin or Bcl-2.
In exemplary embodiments of the present invention, the selected DNA segment may also comprises a second coding region encoding a selected protein. In particular embodiments of the present invention, the first coding region may encodes a selected non-marker protein and the second coding region encodes a selected marker protein.
In embodiments wherein expression of the selected DNA segment is desired, the DNA segment is operatively positioned under the control of a promoter, exemplified by, but not limited to, the CMV promoter, the October-4 promoter or the pgk promoter, that expresses the DNA segment in the primordial germ cells. In other embodiments of the present invention, the selected DNA segment is operatively positioned in reverse orientation under the control of the promoter, wherein the promoter directs the expression of an antisense product.
In still other embodiments of the instant invention, the DNA segment comprises two selected DNA regions that flank the coding region, thereby directing the homologous recombination of the coding region into the genomic DNA of a non-rodent animal species. In more preferred embodiments, the selected DNA regions correspond to distinct sequences in the genomic DNA of the non-rodent animal species. In exemplary embodiments, the isolated DNA regions correspond to the October-4 gene, or regions that flank the October-4 gene.
In still other embodiments of the present invention, the DNA segment comprises two selected DNA sequences that flank the DNA segment and allow for excision of the DNA segment under appropriate conditions. In particularly preferred embodiments, the DNA sequences are loxP sites.
In certain preferred methods of the present invention, the non-rodent animal species is bovine, ovine, porcine, caprine or equine. In other preferred methods, the non-rodent animal is a buffalo or a rabbit.
The present invention thus provides primordial germ cells of a non-rodent animal species that contain a selected DNA segment that may be prepared by a process comprising the steps of introducing the selected DNA segment into a composition comprising isolated primordial germ cells from a non-rodent animal species to obtain candidate primordial germ cells of the non-rodent animal species that contain the selected DNA segment, and plating the candidate primordial germ cells of the non-rodent animal species that contain the selected DNA segment on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain the primordial germ cells of the non-rodent animal species that contain the selected DNA segment.
The invention also provides primordial germ cells of a non-rodent animal species that contain a selected DNA segment that may be prepared by a process comprising the steps of plating a composition comprising primordial germ cells on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, introducing the selected DNA segment into the composition comprising isolated primordial germ cells from a non-rodent animal species to obtain candidate primordial germ cells of the non-rodent animal species that contain the selected DNA segment, and plating the candidate primordial germ cells on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain the primordial germ cells of the non-rodent animal species that contain the selected DNA segment
In further aspects of the present invention, primordial germ cells of a non-rodent animal species that contain a selected DNA segment are provided that may be prepared by a process comprising the steps of plating a composition comprising primordial germ cells on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, introducing the selected DNA segment into the composition comprising isolated primordial germ cells from a non-rodent animal species to obtain candidate primordial germ cells of the non-rodent animal species that contain the selected DNA segment, plating the candidate transformed primordial germ cells on feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, and screening the candidate primordial germ cells of a non-rodent animal species for the presence of the selected DNA segment, to obtain the primordial germ cells of the non-rodent animal species that contain the selected DNA segment.
The present invention also provides a method of producing a transgenic non-rodent animal comprising introducing a selected DNA segment into a composition comprising primordial germ cells from said non-rodent animal to obtain candidate primordial germ cells that contain said selected DNA segment, plating said candidate primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain said primordial germ cells of said non-rodent animal that contain said selected DNA segment, and generating a transgenic non-rodent animal from said primordial germ cells of a non-rodent animal that contain said selected DNA segment, wherein said selected DNA segment is contained and expressed in somatic and germ cells of said non-rodent animal.
The present invention additionally provides a method of producing a transgenic pig comprising introducing a selected DNA segment into a composition comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment, plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain porcine primordial germ cells of said non-rodent animal that contain said selected DNA segment, and generating a transgenic pig from said primordial germ cells that contain said selected DNA segment, wherein said selected DNA segment is contained and expressed in somatic and germ cells of said transgenic pig.
The present invention further provides a method of producing a transgenic non-rodent animal comprising plating a composition comprising primordial germ cells on feeder cells, said feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, introducing a selected DNA segment into said composition comprising primordial germ cells from said non-rodent animal to obtain candidate primordial germ cells that contain said selected DNA segment, plating said candidate primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain said primordial germ cells of said non-rodent animal that contain said selected DNA segment, and generating a transgenic non-rodent animal from said primordial germ cells of a non-rodent animal that contain said selected DNA segment, wherein said selected DNA segment is contained and expressed in somatic and germ cells of said non-rodent animal.
Additionally, the present invention provides a method of producing a transgenic non-rodent animal comprising introducing a selected DNA segment into a composition comprising primordial germ cells from said non-rodent animal to obtain candidate primordial germ cells that contain said selected DNA segment, plating said candidate primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 1.5xc3x97105 cells/cm5 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, screening said candidate primordial germ cells for said selected DNA segment, to obtain said primordial germ cells of said non-rodent -animal that contain said selected DNA segment, and generating a transgenic non-rodent animal from said primordial germ cells of a non-rodent animal that contain said selected DNA segment, wherein said selected DNA segment is contained and expressed in somatic and germ cells of said non-rodent animal.
In certain embodiments, the composition comprising primordial germ cells contains cultured cells from a primordial germ cell-derived cell line.
In particular embodiments of the present invention, the transgenic non-rodent animal is generated by a method comprising injecting the primordial germ cells of the non-rodent animal species that contain said selected DNA segment into a blastocyst from said non-rodent animal species. In certain aspects, the transgenic non-rodent animal is generated by a method comprising injecting the primordial germ cells of the non-rodent animal species that contain the selected DNA segment into a blastocyst from the non-rodent animal species, transferring the blastocyst into a synchronized recipient female of the non-rodent animal species to produce a pregnant non-rodent animal, and allowing gestation in the pregnant non-rodent animal to proceed for a period of time sufficient to allow the development of a viable transgenic non-rodent animal. In further embodiments, the viable transgenic non-rodent animal is obtained by natural birth, while in other embodiments, the viable transgenic non-rodent animal is obtained by surgically removing the viable transgenic non-rodent animal from the recipient female.
In other aspects of the present invention, the transgenic non-rodent animal is generated by a method comprising isolating a nucleus from the primordial germ cells of the non-rodent animal that contain the selected DNA segment and injecting the nucleus into an enucleated oocyte from the non-rodent animal. In particular embodiments, the transgenic non-rodent animal is generated by a method comprising, isolating a nucleus from the primordial germ cells of the non-rodent animal that contain the selected DNA segment and injecting the nucleus into an enucleated oocyte from said non-rodent animal species, transferring the oocyte into a synchronized recipient female of the non-rodent animal species to produce a pregnant non-rodent animal, and allowing gestation in the pregnant non-rodent animal to proceed for a period of time sufficient to allow the development of a viable transgenic non-rodent animal.
In still other embodiments of the present invention, the transgenic non-rodent animal is generated by a method comprising aggregating the primordial germ cells of the non-rodent animal species that contain the selected DNA segment with an early stage embryo of the non-rodent animal species. In certain aspects, the transgenic non-rodent animal is generated by a method comprising aggregating the primordial germ cells of the non-rodent animal species that contain the selected DNA segment with an early stage embryo of the non-rodent animal species, transferring the embryo into a synchronized recipient female of the non-rodent animal species to produce a pregnant non-rodent animal, and allowing gestation in the pregnant non-rodent animal to proceed for a period of time sufficient to allow the development of a viable transgenic non-rodent animal.
The present invention also provides a transgenic non-rodent animal that may be prepared by a process comprising the steps of introducing a selected DNA segment into a composition comprising primordial germ cells from the non-rodent animal to obtain candidate primordial germ cells that contain the selected DNA segment, plating the candidate primordial germ cells that contain the selected DNA segment on feeder cells, the feeder cells at a density of between about 1.5xc3x97105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain the primordial germ cells of the non-rodent animal species that contain the selected DNA segment, and generating a transgenic non-rodent animal from the primordial germ cells of the non-rodent animal that contain the selected DNA segment, wherein the selected DNA segment is contained and expressed in somatic and germ cells of the non-rodent animal. In particular aspects of the present invention, the transgenic non-rodent animal is a cow, sheep, pig, horse, buffalo, rabbit or a goat.
The invention also provides a composition comprising primordial germ cells from a non-rodent animal species, feeder cells sufficient to achieve a density of between about 1.5xc3x97105 and about 106 feeder cells/cm2, and basic fibroblast growth factor in an amount effective to promote the growth and continued proliferation of said primordial germ cells.
In certain aspects of the invention, the primordial germ cells comprise at least a first exogenous DNA segment. In other aspects, the feeder cells are STO cells. In particular embodiments of the invention, the composition may further comprise one or more of uteroferrin, xcex12-macroglobulin, leukemia inhibitory factor, soluble stem cell factor, amino acids non-essential to the non-rodent animal species contemplated for use, L-glutamine, xcex2-mercaptoethanol, Dulbecco""s modified Eagle""s media and/or Ham""s F10 media in an amount effective to promote the growth and continued proliferation of said primordial germ cells.
In further aspects of the present invention, the primordial germ cell is a bovine, ovine, porcine, caprine, equine, buffalo or rabbit primordial germ cell. In preferred embodiments, the primordial germ cell is porcine primordial germ cell.
The invention also provides for the use of any of the disclosed compositions in the preparation of a primordial germ cell-derived cell line. Thus, the instant compositions are contemplated for use in the preparation of a primordial germ cell-derived cell line. Further, the invention provides for the use of any of the disclosed compositions comprising an exogenous DNA segment in the preparation of a transgenic non-rodent animal. Therefore, the compositions comprising an exogenous DNA segment of the present invention are contemplated for use in the preparation of a transgenic non-rodent animal.
The present invention also provides a variety of kits for use in the practice of certain of the methods disclosed and/or claimed herein. The invention provides for the use of any of the disclosed compositions comprising primordial germ cells in the preparation of a kit. Thus, any of the primordial germ cell compositions are contemplated for use in the preparation of a kit. In particular aspects, the kit may comprise, in suitable container means, primordial germ cells from a non-rodent animal species, feeder cells sufficient to achieve a density of between about 1.5xc3x97105 and about 106 feeder cells/cm2, and basic fibroblast growth factor in an amount effective to promote the growth and continued proliferation of said primordial germ cells.